Study On Antihypertension And Improving Vascular Dysfunction Effects Of Soybean Protein-derived Peptides

As the most common cardiovascular disease,hypertension has become one of the major risk factors for human mortality worldwide.Studies have shown that vascular dysfunction is directly related to the occurrence and development of hypertension.Most clinical drug treatments require lifelong use and have obvious side effects.Therefore,it is a hotspot in the field of functional food that exploring active components from safe and green food-derived biological resources to help improve vascular function and control blood pressure.Bioactive peptides are mainly derived from food-derived protein resources.They are widely used in biomedicine,food processing,nutrition and health care because of various activities about improving memory,anti-hypertension,and reducing blood lipids and blood glucose.Soy protein,egg white protein and casein are the main protein resources for the preparation of antihypertensive peptides by enzymatic hydrolysis.However,there are few reports on the improvement of vascular dysfunction in hypertension by three protein-derived active peptides,especially for extracellular vesicles（EVs）-mediated regulation of vascular dysfunction.Therefore,this study investigated the effects of three protein-derived hydrolysates on vasodilation and the regulation of soybean protein-derived vasoactive peptides on hypertension.（1）Soybean protein,egg white protein and casein were prepared to the hydrolysates by pepsin,flavourzyme,neutrase,papain,and alcalase under the optimum conditions.The protein recovery rate,degree of hydrolysis,and molecular weight distribution of each hydrolysate were analyzed.The effects of each hydrolysate on vasodilation and NO production were detected by isolated aortic rings and human umbilical vein endothelial cells（HUVEC）.The alcalase-hydrolysate and flavourzyme-hydrolysate from soybean protein have a high enzymatic hydrolysis efficiency and significant relaxation on the isolated aortic rings.The alcalase and other four kinds of protease were compounded to prepare the combination hydrolysates of soybean protein.The combination of alcalase and flavourzyme hydrolysates had highest protein recovery rate,degree of hydrolysis and low molecular weight components.It significantly promoted the relaxation of aortic ring in an endothelium-dependent manner,and increased the production of NO in HUVEC.Therefore,soybean protein-derived vasodilator peptide（SVP）can be prepared from combination of alcalase and flavourzyme hydrolysates.（2）The co-culture model of HUVECs and VSMCs was constructed to explore effects of Ang II-induced VSMC-derived EVs(EV_vsmc-A)on HUVEC,and the improvement of SVP on EVs-mediated vascular endothelial dysfunction.The EVs samples were identified as having a typical"cup-like bilayer membrane structure"with a particle size<300 nm,and 1×10⁸particles/m L.The specific biomarkers CD81,CD63,and Tsg101 were expressed.The EV_vsmc-A was able to internalize into HUVECs and promoted the production of ROS,IL-6 and IL-1b.The EV_vsmc-A induced the proliferation and migration of HUVECs,and increased the adhesion to U937 monocytes by upregulating the expression of adhesion molecules ICAM-1,VCAM-1,and E-selectin.The SVP could significantly inhibit EV_vsmc-A-induced oxidative stress,inflammatory,and monocyte adhesion of HUVEC.The EV_vsmc-A lysates were treated with RNase and/or Protease,the lysates containing RNAs retained the damaging effects on HUVECs,that suggesting the RNA may be the vital fraction in the EVs.（3）Differential analysis of miRNAs loaded in normal VSMC-derived EVs(EV_vsmc-C)and EV_vsmc-A was performed through miRNA high-throughput sequencing,and verified by q PCR and western blot.The results showed that EV_vsmc-A had 23 miRNA genes with significantly different expression compared with EV_vsmc-C,of which 8 genes were significantly down-regulated and 15 genes were significantly up-regulated.The target gene enrichment analysis of differential miRNAs showed that AMPK,Wnt and m TOR signaling pathways should be related to miRNA regulation.EV_vsmc-A significantly activated the expression of key genes in AMPK,Wnt and m TOR signaling pathways of HUVECs.It was verified that miR-19b was most significantly enriched in EV_vsmc-A,and the SVP could significantly inhibit the enrichment of miR-19b in EVs.EVv_smc-A with miR-19b enrichment could down-regulate the expression of deubiquitinase CYLD of HUVEC,which induced the accumulation of TRAF6（a substrate of CYLD）in HUVEC,and then activated the NF-k B signaling pathway and increased IL-6 and IL-1b of HUVEC.The SVP pre-treatment could inhibit the down-regulation of CYLD in EV_vsmc-A-induced HUVEC,and maintain the normal degradation process of TRAF6,thereby reducing the production of IL-6 and IL-1b in HUVECs.The SVP inhibited the activation of CYLD/NF-k B signaling pathway in HUVECs by reducing the overexpression of miR-19b in EVs from Ang II-induced VSMC.（4）The in vivo experiments showed that both SVP and captopril gavage for 4 weeks could significantly reduce the systolic blood pressure（SBP）and diastolic blood pressure（DBP）of SHRs.The SBP of SVP high-dose group（500 mg/kg·BW）decreased about 70 mm Hg.The SVP significantly increased the serum enzyme activity of SOD,GSH-Px,and the serum content of GSH SHRs,and reduced the serum content of MDA.In addition,SVP significantly improved the inflammatory response of SHRs by reducing serum IL-6,IL-1b,and TNF-a levels.Further studies found that SVP could reduce serum Ang II,renin,ET-1,and aldosterone,and increase the NO content in serum.Pathological results showed that SVP could significantly reduce renal inflammatory cell infiltration and improve aortic remodeling in SHRs.The q PCR showed that m RNA expressions of inflammatory factors IL-6,IL-1b,and TNF-a were significantly down-regulated in the aorta of SHRs with SVP gavage.The SVP up-regulated the expression of CYLD and down-regulated the expression of TRAF6,p-Ik Ba,and NF-k B p65 in the aorta of SHRs,thereby inhibiting the activation of NF-k B signaling pathway.（5）Compared with normotensive WKYs-derived serum EVs(EV_WKY),the miR-19b is abundantly expressed in SHRs-derived serum EVs(EV_SHR).The EV_SHR transplantation significantly reduced the expression of CYLD in aorta of SHRs,and induced the up-regulation of TRAF6,p-Ik Ba,and NF-k B p65 expressions in aorta of SHRs.The inflammation-related NF-k B signaling pathway was activated;The expression of miR-19b in the serum EVs from SHRs with SVP gavage was significantly decreased.Both EV_SVP and EV_WKY transplantation could reduce the blood pressure and improve the degree of aortic wall thickening and fibrosis in SHRs.EV_SVP transplantation reduced the serum levels of inflammatory factors IL-6,IL-1b,and TNF-a in SHRs,and up-regulated the expression of CYLD in the aorta of SHRs.Those effects decreased TRAF6,p-Ik Ba,and NF-k B p65 in the aorta of SHRs,thereby inhibiting the activation of NF-k B signaling pathway.Those results indicated that EV_SVP might exert a physiological regulation in the circulatory system of SHRs by improving aortic inflammatory response and vascular cell phenotype transformation.（6）There were 7977 peptide sequences were identified from the hydrolysate SVP by nano-HPLC-MS/MS,and their lengths ranged from 2 to 33,of which 6980 were 10 peptides and below.The amino acid composition characteristics of 6980 small peptides showed the small peptide N-terminal Trp（W）,Thr（T）,Glu（E）and Lys（K）accounted for a higher proportion.Among the 6980 small peptides,there were 1265 peptides with a Peptide Ranker score>0.8,there were 877 peptides with a CPPPred score>0.5.There were 68 small peptide sequences in the intersection of the two groups of peptide sequences,and they were considered to have high biological activity and cell membrane permeability.There were 29 tetrapeptides,27pentapeptides,9 hexapeptides and 3 heptapeptides in the 68 small peptides.The Allergen FP v.1.0 and Toxin Pred database were used to predict the allergen and biotoxicity of 68 peptides.It was found that 68 peptides had no biological toxicity,and 47 peptide sequences had no potential allergen.The pure peptides ALWW,WLRL,LWLP,MLLW,WGPRL and WKLPM were synthesized to verify bioactivities.The results showed that WLRL and WGPRL could promote aortic ring relaxation in a dose-dependent manner,and significantly reduce the overproduction of ROS,IL-6,IL-1b,and TNF-a in EV_vsmc-A-induced HUVEC.Meanwhile,peptides WLRL and WGPRL inhibited the activation of NF-k B signaling pathway by increasing the expression of CYLD in EV_vsmc-A-induced HUVEC,thereby reducing the expression of Ik Ba,and NF-k B p65.